LETTER
doi:10.1038/nature11071
Melanoma genome sequencing reveals frequent
PREX2 mutations
Michael F. Berger1{*, Eran Hodis1*, Timothy P. Heffernan2{*, Yonathan Lissanu Deribe2{*, Michael S. Lawrence1,
Alexei Protopopov2{, Elena Ivanova2, Ian R. Watson2{, Elizabeth Nickerson1, Papia Ghosh2, Hailei Zhang2, Rhamy Zeid2,
Xiaojia Ren2, Kristian Cibulskis1, Andrey Y. Sivachenko1, Nikhil Wagle2,3, Antje Sucker4, Carrie Sougnez1, Robert Onofrio1,
Lauren Ambrogio1, Daniel Auclair1, Timothy Fennell1, Scott L. Carter1, Yotam Drier5, Petar Stojanov1, Meredith A. Singer2{,
Douglas Voet1, Rui Jing1, Gordon Saksena1, Jordi Barretina1, Alex H. Ramos1,3, Trevor J. Pugh1,2,3, Nicolas Stransky1,
Melissa Parkin1, Wendy Winckler1, Scott Mahan1, Kristin Ardlie1, Jennifer Baldwin1, Jennifer Wargo6, Dirk Schadendorf4,
Matthew Meyerson1,2,3,7, Stacey B. Gabriel1, Todd R. Golub1,7,8,9, Stephan N. Wagner10, Eric S. Lander1,11*, Gad Getz1*,
Lynda Chin1,2,3{* & Levi A. Garraway1,2,3,7*
Melanoma is notable for its metastatic propensity, lethality in the
advanced setting and association with ultraviolet exposure early in
life1. To obtain a comprehensive genomic view of melanoma in
humans, we sequenced the genomes of 25 metastatic melanomas
and matched germline DNA. A wide range of point mutation rates
was observed: lowest in melanomas whose primaries arose on nonultraviolet-exposed hairless skin of the extremities (3 and 14 per
megabase (Mb) of genome), intermediate in those originating from
hair-bearing skin of the trunk (5–55 per Mb), and highest in a
patient with a documented history of chronic sun exposure (111
per Mb). Analysis of whole-genome sequence data identified
PREX2 (phosphatidylinositol-3,4,5-trisphosphate-dependent Rac
exchange factor 2)—a PTEN-interacting protein and negative regulator of PTEN in breast cancer2—as a significantly mutated gene
with a mutation frequency of approximately 14% in an independent
extension cohort of 107 human melanomas. PREX2 mutations
are biologically relevant, as ectopic expression of mutant PREX2
accelerated tumour formation of immortalized human melanocytes
in vivo. Thus, whole-genome sequencing of human melanoma
tumours revealed genomic evidence of ultraviolet pathogenesis
and discovered a new recurrently mutated gene in melanoma.
To gain a comprehensive view of the genomic landscape in human
melanoma tumours, we sequenced the genomes of 25 metastatic
melanomas and peripheral blood obtained from the same patients
(Supplementary Table 1). Two tumours (ME015 and ME032) were
metastases from cutaneous melanomas arising on glabrous (that is,
hairless) skin of the extremities, representing the acral subtype. The
other tumours were primarily metastases from melanomas originating
on hair-bearing skin of the trunk (the most common clinical subtype).
Further, ME009 represented a metastasis from a primary melanoma of
a patient with a clinical history of chronic ultraviolet exposure.
We obtained 59-fold mean haploid genome coverage for tumour
DNA and 32-fold for normal DNA (Supplementary Table 2). On
average, 78,775 somatic base substitutions per tumour were identified,
consistent with prior reports3,4 (Supplementary Table 3). This corresponded to an average mutation rate of 30 per Mb. However, the
mutation rate varied by nearly two orders of magnitude across the
25 tumours (Fig. 1). The acral melanomas showed mutation rates
comparable to other solid tumour types (3 and 14 mutations per
Mb)5,6, whereas melanomas from the trunk harboured substantially
more mutations, in agreement with previous studies3,7,8. In particular,
sample ME009 exhibited a striking rate of 111 somatic mutations per
Mb, consistent with a history of chronic sun exposure.
In tumours with elevated mutation rates, most nucleotide substitutions were C R T or G R A transitions consistent with ultraviolet
irradiation9. The variations in mutation rate correlated with differences in the ultraviolet mutational signature. For example, 93% of
substitutions in ME009 but only 36% in acral melanoma ME015 were
C R T transitions (Fig. 1); these tumours contained the highest and
lowest base mutation rates, respectively (111 and 3 mutations per Mb).
Interestingly, the acral tumour ME032 also showed a discernible
enrichment of ultraviolet-associated mutations (Fig. 1). Thus, genome
sequencing readily confirmed the contribution of sun exposure in
melanoma aetiology.
In agreement with prior studies7,9, we detected an overall enrichment for dipyrimidines at C R T transitions. Analysis of intragenic
C R T mutations yielded a significant bias against such mutations on
the transcribed strand for most melanomas, consistent with transcription-coupled repair (Supplementary Fig. 1)3,7,10. Most commonly,
C R T mutations occurred at the 39 base of a pyrimidine dinucleotide
(CpC or TpC; Supplementary Fig. 2). In contrast, the C R T mutations
in sample ME009 (with hypermutation and chronic sun exposure
history) more often occurred at the 59 base of a pyrimidine dinucleotide.
As expected, the acral tumour ME015 exhibited mutation patterns
observed in non-ultraviolet-associated tumour types11, such as an
increased mutation rate at CpG dinucleotides relative to their overall
genome-wide frequency (Supplementary Fig. 2). These different mutational signatures suggest a complex mechanism of ultraviolet mutagenesis
across the clinical spectrum of melanoma, probably reflecting distinct
histories of environmental exposures and cutaneous biology.
We detected 9,653 missense, nonsense or splice site mutations in
5,712 genes (out of a total of 14,680 coding mutations; Supplementary
Tables 4 and 5), with an estimated specificity of 95% (Supplementary
Methods). A mutation of BRAF, BRAFV600E, was present in 16 of 25
tumours (64%), including the acral melanoma ME015. NRAS was
mutated in 9 of 25 tumours (36%) in a mutually exclusive fashion with
1
The Broad Institute of Harvard and MIT, Cambridge, Massachusetts 02142, USA. 2Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts 02115, USA. 3Harvard Medical
School, Boston, Massachusetts 02115, USA. 4Department of Dermatology, University Hospital Essen, D-45122 Essen, Germany. 5Department of Physics of Complex Systems, Weizmann Institute of
Science, Rehovot 76100, Israel. 6Department of Surgery, Massachusetts General Hospital, Boston, Massachusetts 02114, USA. 7Center for Cancer Genome Discovery, Dana-Farber Cancer Institute, Boston,
Massachusetts 02115, USA. 8Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts 02115, USA. 9Howard Hughes Medical Institute, Chevy Chase, Maryland 20815, USA.
10
Division of Immunology, Allergy and Infectious Diseases, Department of Dermatology, Medical University of Vienna and CeMM-Research Center for Molecular Medicine of the Austrian Academy of
Sciences, 1090 Vienna, Austria. 11Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, Massachusetts 02142, USA. {Present address: Department of Pathology, Memorial SloanKettering Cancer Center, New York, New York 10065, USA (M.F.B.); Department of Genomic Medicine, Institute for Applied Cancer Science, The University of Texas MD Anderson Cancer Center, Houston,
Texas 77030, USA (T.P.H., Y.L.D., A.P., I.R.W., M.A.S., L.C.).
*These authors contributed equally to this work.
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©2012 Macmillan Publishers Limited. All rights reserved
100
111
No. of mutations per Mb
LETTER RESEARCH
80
60
40
20
0
BRAF
NRAS
64%
36%
100
5
15 10
0
No. of mutations
80
60
40
ME009
ME044
ME100L
ME049
ME043
ME002
ME024
ME029
ME020
ME050
ME018
ME041
ME011
ME016
ME037
ME001
ME030
ME035
ME048
ME045
ME012
ME032
ME021
ME007
ME015
20
Percentage
Indels and other muts
Transversions
A→G
(A/C/G)pC→(A/C/G)pT
TpC→TpT
0
Figure 1 | Elevated mutation rates and spectra indicative of ultraviolet
radiation damage. Top bar plot shows somatic mutation rate of 25 sequenced
melanoma genomes, in decreasing order. Middle matrix indicates BRAF and
NRAS somatic mutation status, with left-adjacent bar plot indicating total
number of mutations in each oncogene as well as percent frequency. Bottom
plot displays each tumour’s somatic mutation spectrum as a percentage of all
mutations (right axis). Tumour sample names are indicated at the bottom of the
figure, with acral melanomas in red.
BRAF, with the exception of one non-canonical substitution
(NRAST50I) in the hypermutated sample ME009. We also identified 6
insertions and 34 deletions in protein coding exons (Supplementary
Table 6), including a 21-base-pair (bp) in-frame deletion involving exon
11 of the KIT oncogene in the acral tumour ME032 (Supplementary
Fig. 3). KIT mutations occur in 15% of acral and mucosal melanomas12,
and melanoma patients with activating KIT mutations in exon 11 have
demonstrated marked responses to imatinib treatment13.
We identified an average of 97 structural rearrangements per
melanoma genome (range: 6–420) (Supplementary Table 7). In addition to displaying a wide range of rearrangement frequencies, the
proportion of intrachromosomal and interchromosomal rearrangements varied widely across genomes. ME029, which harboured the
largest number of rearrangements (420), contained only 8 interchromosomal events (Fig. 2a). In contrast, ME020 and ME035 contained 95 and 90 interchromosomal rearrangements, respectively
(Fig. 2a). In both cases, the vast majority of interchromosomal rearrangements were restricted to two chromosomes. This pattern is reminiscent
of chromothripsis14, a process involving catastrophic chromosome
breakage that has been observed in several tumour types15,16.
106 genes harboured chromosomal rearrangements in two or more
samples (Supplementary Table 8). Many recurrently rearranged loci
contain large genes or reside at known or suspected fragile sites17;
examples include FHIT (six tumours), MACROD2 (five tumours)
and CSMD1 (four tumours). On the other hand, several known cancer
genes were also recurrently rearranged, including the PTEN tumour
suppressor (four tumours) and MAGI2 (three tumours), which
encodes a protein known to bind and stabilize PTEN. MAGI2 was also
found disrupted in recent whole-genome studies of prostate cancer18
and a melanoma cell line7. Rearrangements involving the 59 untranslated region of the ataxin 2-binding protein 1 gene (A2BP1) were
observed in 4 tumours. A2BP1 encodes an RNA binding protein whose
genetic disruption has been linked to spinocerebellar ataxia and other
neurodegenerative diseases. A2BP1 undergoes complex splicing regulation in the central nervous system and other tissues19; in melanoma,
these rearrangements may disrupt a known A2BP1 splice isoform or
enable a de novo splicing product. Together, these results suggest that
chromosomal rearrangements may contribute importantly to melanoma
genesis or progression.
An acral melanoma (ME032) harboured the second-largest number
of total rearrangements (314; Fig. 2a). We employed high-throughput
PCR followed by massively parallel sequencing to successfully validate
177 of 182 events tested in this sample, confirming its high rate of
rearrangement. The elevated frequency of genomic rearrangements in
acral melanomas has been reported previously20. In comparison,
ME032 exhibited one of the lowest base-pair mutation rates of the
melanomas examined (22nd out of 25 samples), suggesting that different tumours might preferentially enact alternative mechanisms of
genomic alteration to drive tumorigenesis.
As noted above, many rearrangements in ME032 involved multiple
breakpoints within a narrow genomic interval. One such event disrupted the ETV1 locus. We previously demonstrated an oncogenic role
for ETV1 in melanoma, whose dysregulated expression was associated
with upregulation of microphthalmia-associated transcription factor
(MITF)21, the master melanocyte transcriptional regulator and a
melanoma lineage survival oncogene22. We validated six distinct
rearrangements (four interchromosomal translocations) in ME032
involving breakpoints within ETV1 introns (Fig. 2b). These events join
regions of ETV1 to distal loci on chromosomes 8, 9, 11 and 15. In
support of their possible functional relevance, these rearrangements
were associated with high-level ETV1 amplification in this tumour.
A second complex rearrangement involved the PREX2 locus.
PREX2 encodes a phosphatidylinositol 3,4,5-trisphosphate RAC
exchange factor recently shown to interact with the PTEN tumour
suppressor and modulate its function2. We validated nine somatic
rearrangements in the vicinity of PREX2 (six interchromosomal translocations), including five with intronic breakpoints (Fig. 2c, Supplementary Fig. 4). One event joined specific intronic regions of
PREX2 and ETV1. Like ETV1, PREX2 is highly amplified in this
tumour, as verified by FISH (fluorescence in situ hybridization) analysis
(Fig. 2d, Supplementary Fig. 5). The presence of these complex structural rearrangements in addition to amplification may indicate multiple
mechanisms of PREX2 dysregulation in melanoma. More generally,
these findings raised the possibility that sites of complex rearrangement
might denote genes of functional importance in melanoma.
Next, we calculated the mutational significance of each gene based
on the number of mutations detected, gene length and background
mutation rates (Table 1, Supplementary Table 9) (see Methods).
Eleven genes were found to be significantly mutated across the 25
samples (Q , 0.01, where Q is the false discovery rate adjusted
P-value). As expected, the two most significant genes were BRAF
and NRAS, mutated in 16 and 9 samples, respectively. Interestingly,
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©2012 Macmillan Publishers Limited. All rights reserved
RESEARCH LETTER
a
1
1
2
2
9
11
10
9
11
11
10
2
2
9
8
10
6
7
8
8
8
9
5
12
7
11
4
14
11 Mb deletion (intron of LFNG)
To chr. 11 (5’ UTR of ODZ4)
To chr. 15 (intron of RASGRP1)
To chr. 9 (intron of SUSD1)
5 kb inversion
To chr. 8 (intron of PREX2)
3′
c
3
13
6
12
7
7
10
20
19
18
17
16
15
5
13
X
4
Y
3
14
6
12
b ETV1 (sample ME032)
20
19
18
17
16
15
22
21
5
ME032
Y
4
13
6
12
3
14
5
13
22
21
14
20
19
18
17
16
15
X
4
Y
22
21
3
ME035
X
Y
22
21
X
20
19
18
17
16
15
1
ME020
1
ME029
5′
PREX2 (sample ME032)
To chr. 7 (intron of ETV1)
To chr. 7 (near CARD11)
To chr. 7 (near PER4)
5 Mb tandem duplication (intron of STAU2)
640 kb tandem duplication (near CPA6)
4 Mb inversion (near TRPA1)
To chr. 7 (near ICA1)
To chr. 7 (near
TTYH3)
E824*
To chr. 11 (near C11orf30)
5′
3′
Amplifications
d
PREX2
Figure 2 | Hubs of rearrangement breakpoints affect known and putative
oncogenes. a, Circos plots representing four melanoma genomes with notable
structural alterations. Interchromosomal and intrachromosomal
rearrangements are shown in purple and green, respectively. b, Location of
breakpoints associated with ETV1 in melanoma ME032. c, Location of
breakpoints associated with PREX2 in melanoma ME032. The red arrow
indicates a premature stop codon (E824*). All rearrangements in ETV1 and
PREX2 were validated by high-throughput PCR and deep sequencing.
d, Confirmation of high-level amplification and rearrangement of PREX2 in
ME032 by dual-colour break-apart FISH. The assignment of red and green
FISH probes to the PREX2 gene region is delineated as bars. Lack of colocalization of red and green probes is indicative of break-apart.
PREX2 scored as one of the top significant genes (Table 1).
Furthermore, four samples harboured nonsense truncation mutations
in PREX2, more than any of the other genes identified as statistically
significant in this analysis. PREX2 mutations have occasionally been
reported in colon, lung and pancreatic cancer23, albeit at low frequencies. Here, we detected 13 non-synonymous point mutations in
PREX2—including 4 nonsense mutations—and 1 synonymous mutation, with 11 of 25 melanomas harbouring at least 1 non-synonymous
mutation. The mutations were distributed throughout the entire
length of PREX2 (Fig. 3a, green circles), and 13 of 14 mutations were
non-synonymous, suggestive of positive selection. An analysis of the
mutant allele frequencies and estimated tumour purities indicates that
at least two mutations are homozygous. One melanoma, ME018,
harbours three missense mutations, two of which (I534M and
G1581R) appear to co-occur on a single allele based on their observed
mutation frequencies. Notably, a PREX2 nonsense mutation was
detected in ME032, in addition to the rearrangements and amplification of this locus present in this tumour (Fig. 2c). This PREX2
mutation was truncating (E824*), removing the carboxy-terminal
region with homology to an inositol phosphatase domain. Based on
the allele frequency of this mutation, we infer that it occurs on the
non-amplified allele. Taken together, whole-genome sequencing of
this 25-sample discovery cohort identified PREX2 as a candidate melanoma gene whose amplifications, rearrangements or mutations
appeared to undergo positive selection in human melanoma genesis.
To determine the prevalence of PREX2 mutations in melanoma, we
performed bidirectional capillary sequencing in an extension cohort of
107 tumour/normal pairs, comprising 45 tumours and 62 short-term
cultures collected from multiple institutions and geographic regions
(Supplementary Table 10). We identified 23 somatic base pair mutations and one frame-shift insertion in PREX2 in this cohort (Fig. 3a;
Supplementary Table 11), 15 of which represented non-synonymous
changes. We therefore inferred a 14% frequency of non-synonymous
PREX2 mutations in this melanoma cohort.
Discrepant non-synonymous:synonymous ratios were observed
between the tumour samples and short-term cultures in the extension
cohort. In line with results from the discovery cohort, 100% of PREX2
mutations detected across 45 tumour samples were non-synonymous
in nature (n 5 4), consistent with positive selection. In contrast, only
55% of the sequence mutations found in the 64 short-term cultures
were non-synonymous (a ratio of 11:9). Conceivably, these findings
may indicate that subsets of melanoma cells capable of robust growth
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©2012 Macmillan Publishers Limited. All rights reserved
LETTER RESEARCH
Table 1 | Significantly mutated genes in 25 melanoma tumours.
Rank
Gene
Total number of
covered bases
Samples with
non-synonymous mutations
Non-synonymous
mutations
Nonsense
mutations
Synonymous
mutations
P value
Q value
1
2
3
4
5
6
7
8
9
10
11
BRAF*
NRAS
MUC4
PREX2
GOLGA6L6
VCX3B
POTEH
OR2T33
C1orf127
PRG4
MST1
56,520
14,160
77,038
127,041
18,902
7,132
21,545
21,978
53,004
100,212
46,400
12
9
19
11
5
4
5
5
6
8
8
12
9
42
13
6
4
7
5
6
9
12
0
0
0
4
0
0
0
0
0
0
0
0
0
17
1
1
1
1
1
0
1
4
,10215
4 3 10-15
2 3 10211
2 3 1028
6 3 1027
7 3 1027
8 3 1027
9 3 1027
2 3 1026
3 3 1026
5 3 1026
,10211
4 3 10211
2 3 1027
8 3 1025
2 3 1023
2 3 1023
2 3 1023
2 3 1023
3 3 1023
6 3 1023
9 3 1023
* BRAFV600E mutations were detected in four additional samples by exon capture on manual review of Illumina sequencing data (shown in Fig. 1).
genomic evidence for the role of ultraviolet mutagenesis in melanoma,
and identifies several recurrently mutated and rearranged genes not
previously implicated in this malignancy. In particular, we discovered
that PREX2 mutations are both recurrent and functionally consequential
in melanoma biology. Although its precise mechanism(s) of action
remains to be elucidated in melanoma, PREX2 appears to acquire
oncogenic activity through mutations that perturb or inactivate one or
more of its cellular functions. This pattern of mutations may exemplify a
category of cancer genes that is distinct from ‘classic’ oncogenes (often
characterized by highly recurrent gain-of-function mutations) and
tumour suppressors (inactivated by simple loss-of-function alterations).
Instead, (over)expression of certain cancer genes with distributed mutation patterns may promote tumorigenicity either through dominant
negative effects or more subtle dysregulation of normal protein functions.
Cancer genomics has enabled the discovery and rational application
of the first truly effective targeted therapy for metastatic melanoma:
BRAF mutations predict sensitivity to selective RAF inhibitors25–27.
However, the emergence of acquired resistance is rapid and often
driven by other genomic events28. Our genomic exploration of the
melanoma genomes revealed a large number of complex alterations
that probably affect many other genes in addition to PREX2.
Understanding how this spectrum of genomic aberrations contributes
to melanoma genesis and progression should provide new insights into
tumour biology, therapeutic resistance and development of treatment
regimens aimed at durable control of this malignancy.
in vitro may have experienced reduced selective pressure for PREX2
mutations. Alternatively (or in addition), the PREX2 locus may exhibit
an enhanced ‘local’ mutation rate, a by-product of which is the production of variants that undergo positive selection in vivo.
To demonstrate the functional relevance of PREX2 mutations in
melanoma tumorigenesis, we ectopically expressed six representative
mutations (three truncation variants and three non-synonymous point
mutations predicted to carry functional impact24) in TERT-immortalized
human melanocytes engineered to express NRAS(G12D) (PMELNRAS*)21. These melanocytic lines were transplanted into immunodeficient mice alongside control melanocytes expressing either wild-type
PREX2 or GFP (green fluorescent protein). Overexpression of all three
truncated variants as well as a point mutant (G844D) of PREX2 significantly accelerated in vivo tumorigenesis when compared to GFP control
or wild-type PREX2-expressing melanocytes (Fig. 3b, Supplementary Fig. 6). These results therefore affirmed the aforementioned
genomic data suggesting that PREX2 mutations may undergo positive
selection in vivo. Although the spectrum of PREX2 mutations in human
melanoma (Fig. 3a) is reminiscent of inactivating mutations, our
findings suggest that PREX2 somatic mutations generate truncated or
variant proteins that gain oncogenic activity in melanoma cells.
In summary, following recent efforts to characterize whole genomes
from several haematologic and solid tumours, we provide the first (to our
knowledge) high-resolution view of the genomic landscape across a
spectrum of metastatic melanoma tumours. The analysis reveals global
a
0
200
400
600
800
1000
1200
E1356K
A1376V
E1389K
Q1430*
V1441F
A1549V
R1557C
Q1581R
G1196E
S1052F
P948S
T994A
P665L
E730*
S771fs
P775S
P776S
E824*
G844D
I534M
E354D
S413N
E421K
R463C
K278*
E213K
E12*
G106E
PREX2
1400
Sanger
Illumina
DH
PH
DEP1
DEP2
PDZ1
PDZ2
1600
Survival (%)
b
100
80
60
40
20
0
PREX2 WT
GFP
0
2
4
6
Weeks
8
10
100
80
60
40
20
0
100
80
60
40
PREX2 K278* (P = 0.022)
PREX2 E824* (P = 0.018)
20
PREX2 Q1430* (P = 0.0007)
0
10
0
PREX2 WT
0
2
4
6
Weeks
8
Figure 3 | Mutant PREX2 expression promotes melanoma genesis. a, Nonsynonymous sequence mutations detected from Illumina sequencing of 25
melanomas (green) or from capillary sequencing of a validation cohort of 107
additional melanomas (purple). Mutations are dispersed throughout all
annotated structural domains of PREX2. (DH, DBL homology domain;
PH, plekstrin homology domain). The C-terminal half of PREX2 exhibits
PREX2 WT
PREX2 P948S
PREX2 G106E
PREX2 G844D (P = 0.006)
2
4 6
Weeks
8
10
sequence homology to an inositol phosphatase domain. Engineered PREX2
mutants are labelled with red arrowheads. b, Kaplan-Meier curve showing
tumour-free survival of NUDE mice (n 5 10) injected with PMEL-NRAS* cells
expressing GFP, wild-type (left plot), truncated (middle plot) and mutated
(right plot) PREX2 subcutaneously.
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©2012 Macmillan Publishers Limited. All rights reserved
RESEARCH LETTER
METHODS SUMMARY
The complete genomes of 25 metastatic melanomas and patient-matched germline samples were sequenced to approximately 303 and 303 haploid coverage,
respectively, on an Illumina GAIIx sequencer (5 cases), and approximately
653 and 323 haploid coverage, respectively, on an Illumina HiSeq 2000 sequencer
(20 cases) as paired-end 101-nucleotide reads. Read pairs were aligned to the
reference human genome (hg19) using BWA29. Somatic alterations (single base
substitutions, small insertions and deletions, and structural rearrangements) were
identified according to their presence in the tumour genome and absence from the
corresponding normal genome. A subset of rearrangements was validated by PCR
and an independent sequencing technology in order to assess the specificity of the
detection algorithm. Fluorescence in situ hybridization (FISH) was performed to
confirm the high level amplification and rearrangement of PREX2. Significantly
mutated genes were identified by comparing the observed mutations to the
background mutation rates calculated for different sequence context categories
per tumour sample. 40 exons of PREX2 were sequenced by PCR and bidirectional
capillary sequencing in a validation panel of 107 additional melanoma tumours
and short term cultures; mutations were confirmed as somatic by sequencing
matched normal DNA. For gain of function studies, PREX2 mutation constructs
were engineered and introduced to PMEL cell lines by lentiviral transduction. To
assess the oncogenic roles of PREX2 mutants, PMEL-NRAS* cells were injected
subcutaneously into NUDE mice, and tumour growth was measured over time. A
complete description of the materials and methods is provided in Supplementary
Information.
Received 7 September 2010; accepted 9 March 2012.
Published online 9 May 2012.
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Supplementary Information is linked to the online version of the paper at
www.nature.com/nature.
Acknowledgements Illumina sequencing was performed at the Broad Institute and
array-based genomic characterization and functional studies were performed at the
Belfer Institute of DFCI. We are grateful to the Broad Institute Genome Sequencing
Platform, Genome Analysis Platform and Biological Samples Platform. This work was
supported by the National Human Genome Research Institute (S.B.G., E.S.L.), National
Cancer Institute (M.M., L.C.), FWF-Austrian Science Fund (S.N.W.), NIH Director’s New
Innovator Award (L.A.G.), Melanoma Research Alliance (L.A.G., L.C.), Starr Cancer
Consortium (L.A.G.) and the Burroughs-Wellcome Fund (L.A.G.).
Author Contributions M.F.B., E.H., T.P.H. and Y.L.D. are lead authors; E.S.L., G.G., L.C.
and L.A.G. are senior authors. M.F.B. and E.H. performed the genomic analysis of the
whole-genome sequencing data. T.P.H. and Y.L.D. performed the molecular biology
and mouse experiments to interrogate the function of PREX2. E.I., P.G., R.Z. and M.A.S.
participated in the functional experiments. M.S.L. performed genomic analysis of
mutations and rearrangements. A.P. and X.R. performed FISH studies of PREX2. H.Z,
K.C, A.Y.S., T.F., S.L.C., Y.D., P.S., D.V., R.J., G.S., A.H.R., T.J.P. and N.S. provided additional
computational analyses. A.S. and D.S. contributed melanoma short-term cultures for
the extension cohort. I.R.W. contributed clinical information. C.S., R.O., W.W., S.M., D.A.
and K.A. participated in DNA sample processing and quality control. E.N. played a
project management role. M.F.B., M.S.L., R.O., M.P., L.A., W.W., G.G. and L.A.G. validated
candidate rearrangements. J.B. and S.B.G. oversaw the generation of DNA sequence
data. J.B., M.M., S.B.G. and T.R.G contributed to the study design and interpretation of
data. S.N.W. contributed most melanoma tumours for the discovery and extension
cohort and participated in the study design. J.W. and N.W. also contributed tumour
material for the discovery cohort. E.S.L., G.G., L.C. and L.A.G. conceived of and designed
the study and participated in the data analysis and interpretation. M.F.B., T.P.H., L.C.
and L.A.G. wrote the paper.
Author Information All Illumina sequence data are publicly available in dbGaP
(accession number phs000452.v1.p1). Reprints and permissions information is
available at www.nature.com/reprints. This paper is distributed under the terms of the
Creative Commons Attribution-Non-Commercial-Share Alike licence, and is freely
available to all readers at www.nature.com/nature. The authors declare no competing
financial interests. Readers are welcome to comment on the online version of this article
at www.nature.com/nature. Correspondence and requests for materials should be
addressed to L.C. ([email protected]) or L.A.G.
([email protected]).
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